In a unique integrated program of laboratory and clinical investigation we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is elucidating mechanisms by which primary collagen defects cause skeletal fragility and other connective tissue symptoms and applying knowledge gained from our studies to treatment of children with these conditions. Understanding interactions of mutant collagen molecules with normal and non-collagenous components of extracellular matrix will also enhance understanding of normal bone function and may yield insights to more common forms of osteoporosis. We focused on a non-lethal animal model with a classical collagen mutation. This non-lethal knock-in mouse (Brtl), with a glycine substitution mutation in the a1(I) chain, is an excellent model for pharmacological treatment trials, approaches to gene therapy suitable for dominant disorders and studies of OI skeletal matrix. Our clinical studies involve children with types III/IV OI enrolled in age-appropriate treatment protocols forming a longitudinal study group. The OI/EDS Region of the a1(I) Collagen Chain (Cabral, Letocha, Marini, Leikin) Patients with OI/EDS form a distinct subset of OI patients having skeletal fragility as well as characteristics of EDS, severe joint laxity and early onset scoliosis. In OI/EDS children we delineated mutations in the first 90 residues of the helical region of a1(I) chain. These mutations cause abnormal N-propeptide processing, incorporation of pN-collagen into matrix, and decreased diameter of dermal fibrils. This data provides a mechanism for EDS symptoms while the helical changes per se are responsible for bone fragility. The mechanism of their EDS is shared with those with EDS VIIA and B due to absence of N-proteinase cleavage site from a1(I) or a2(I) chain. These assays define a folding region of a1(I) where mutations cause a distinct OI/ED phenotype by altering the triple helical and secondary structure of the N-proteinase cleavage site. Retention of N-propeptide in a substantial proportion of collagen chains limits fibril diameter. The abnormal fibrils may cause joint laxity and paraspinal ligaments directly by reduced resistance to shearing forces, or indirectly, by altering interactions between collagen and other matrix components in the overlap zone of the D-periods. Type I Collagen C-propeptide Mutations (Marini, Barnes, Ashok) Mutations in the C-propeptide of type I collagen are found in some OI patients, the phenotype ranging from lethal to moderately severe. These mutations are of interest as they are located in a region that is cleaved from procollagen before collagen fibril assembly. The mutations per se are not expected in collagen fibrils in tissues. This implies that the pathophysiological mechanism of these mutations differ from mutations in the helical region of alpha chains incorporated into matrix and exert a dominant-negative effect. We identified 4 novel C-propeptide mutations at conserved residues in collagen of children with OI III/IV. All mutations delayed incorporation of alpha1 chains into heterotrimers. A pericellular processing assay suggests a delay in C-propeptide removal from secreted collagens containing these mutations. Mutant collagens are incorporated into fibroblast matrix in culture forming mature cross-links. We compared the intracellular interaction of mutant procollagen molecules with ER chaperones in OI fibroblasts to normal controls and cells with a C-terminal helical mutation using immunofluorescence assays. Results show clear correlation between the presence and type of mutation with subcellular localization pattern of procollagen. Normal procollagen/procollagens with mutations in the carboxyl end of the helical domain show distinct reticular pattern of ER localization with significant immunofluorescence overlap with calnexin, while procollagens with C-propeptide mutations have a diffuse ER localization with an almost complete overlap with the IF pattern of ER chaperones, Hsp-47 and protein disulfide isomerase. Location of the mutation along procollagen chains directs the nature of ER chaperone interactions. Alendronate Treatment of Brtl Mouse (Marini, Uveges, Goldstein, Gronowicz) Bisphosphonates are widely administered to OI children however effects on bone containing abnormal type I collagen have not been directly examined. The Brtl mouse model for type IV OI has a glycine substitution (G349C) knocked-into one COL1A1 allele. We treated Brtl and wild type (wt) offspring of Brtl x CD-1 matings from 2-14 weeks of age with alendronate (0.219 mg/kg/wk, gift of Merck) or saline placebo. Brtl mouse weight and femor length were significantly smaller than wt and unchanged by alendronate. Alendronate treatment increased whole bone density of femurs and lumbar vertebrae in Brtl and wt. Data suggest differences are due to increased bone volume rather than mineralization. Distal femoral bone volume per total volume doubled with treatment due to increased trabecular number. Diaphyseal cortical thickness increased in Brtl and wt femurs. In treated Brtl femurs, geometry reshaped to a more rounded structure. In mechanical testing, alendronate treatment increased femoral stiffness and decreased pre-yield displacement in wt, indicating that treatment is not benign for normal bone. Stiffness, pre-yield displacement, and yield load were unchanged in treated Brtl femora. Alendronate negatively impacts bone quality: 1) predicted material strength and modulus of Brtl and wt bone is decreased; 2) brittleness of treated Brtl femurs was worsened compared to untreated wt; 3) metaphyses of treated Brtl femurs have increased remnants of mineralized cartilage which may increase fracture initiation; and 4) there was a detrimental effect on bone cells. After treatment, BFR/BS, MAR and MS/BS was less than 25% of pretreatment. Percent osteoblast surface decreased. Morphology of Brtl osteoblasts changed from plump cuboidal osteoblasts to an intermediate morphology supporting a toxic effect on the cells. Data suggest limited treatment may be optimal for obtaining improved bone geometry and minimizing detrimental effect of extended treatment on bone quality. Pamidronate Treatment of Children with Types III/IV OI (Letocha, Marini, Gerber, Paul) Uncontrolled trials of bisphosphonates in OI children report increased vertebral bone density and height, improved strength/functional level and decreased fractures and bone pain. We did randomized controlled trial of pamidronate in children with Types III/IV OI. Year 1 was controlled; children in treatment group received pamidronate (10 mg/m2/day for 3 days every 3 months); Children in both treatment and control had PT assessments measuring function, strength and pain. The treatment group received pamidronate for an additional 6-21 months. In the controlled phase, treated patients had significant increase in vertebral BMD z-scores, L1-L4 mid-vertebral height and total vertebral area compared to controls. The treatment group did not have decreased long bone fractures. In extended treatment, DEXA z-scores, vertebral heights and areas did not increase beyond 12-month value. Regarding maximized physical rehabilitation we did not see an additional functional effect from bisphosphonates. In contrast to uncontrolled trials we found no significant changes in ambulation level, lower extremity strength or pain in OI children treated with pamidronate. Within the treatment group, some had a robust response in all measurements while others had increased bone density but not increased area or height. Changes in DEXA z-scores were less than 1 SD to more than 3 SD. This response variability has not been reported and is presumably related to differences in bone matrix caused by underlying collagen mutations.